Lactophenol Cotton Blue (LPCB) Mount: Principle, Procedure, and Fungal Identification

A patient with poorly controlled diabetes develops a rapidly spreading black necrotic lesion involving the palate and periorbital region. CT scan shows sinus involvement. An urgent biopsy is taken. In the mycology laboratory, the tissue is cultured on Sabouraud dextrose agar — and within 48 hours, a rapidly growing fluffy grey-white mould appears. An LPCB mount is prepared.

Under the microscope: broad, non-septate (aseptate) hyphae with irregular branching at wide angles. No structures visible from fruiting heads yet, but the combination of tissue presentation, growth rate, and hyphal morphology is already pointing to Mucorales — mucormycosis, one of the most lethal fungal infections in immunocompromised patients. Treatment decisions cannot wait for species confirmation.

LPCB is the workhorse of the mycology bench — the fastest way to examine fungal morphology in a wet preparation. Understanding what you are looking at under oil immersion is as important as knowing how to prepare the mount.

The lactophenol cotton blue (LPCB) wet mount preparation is the most widely used method of staining and observing fungi and is simple to prepare. The preparation has the following constituents;

Figure: Fig: Aspergillus spp in LPCB Mount

1. Phenol kills fungus.
2. Lactic acid acts as a clearing agent and helps preserve the fungal structures.
3. Cotton blue is an aniline dye that stains the chitin in the fungal cell walls which adds colour to the fungal preparation thereby enhancing and contrasting the structures.
4. Glycerol is a viscous substance that prevents drying of the prepared slide specimen.

Why LPCB is a mounting medium, not a fixative: LPCB preserves and stains fungal structures in their native three-dimensional arrangement — it does not fix or kill organisms immediately on contact (though phenol does kill over time). This is why the preparation should be examined promptly after preparation, and why it is essential to work under a biological safety cabinet with gloves throughout.

Lactophenol cotton blue solution is a mounting medium and staining agent used in the preparation of slides for microscopic examination of fungi. Fungal elements are stained intensely blue. This operation should always be performed under a biological safety hood and gloves should always be worn.

Procedure

Standard Tease Mount

1. Place a drop of 70% alcohol on a clean microscope slide. Material from cultures of filamentous fungi should be removed using a stiff inoculating wire, not the loop used for manipulations with bacteria or yeasts.
2. Flame the wire by holding it upright in the hottest part of the Bunsen flame, just above the blue cone, until the whole length of the wire glows red hot. You must ensure that the inoculating wire has cooled before placing it in a fungal culture – it should have cooled sufficiently after approximately ten seconds.
3. Remove the cap from the culture tube (but do not put it on the bench). Kill any contaminating microorganisms by flaming the neck of the tube.
4. Remove a small amount of the culture. It is often useful to take a little of the agar medium together with the fungus. In any case, the material should be disturbed as little as possible when being transferred to the slide.
5. Flame the neck of the tube once more and replace the cap.
6. Immerse the fungal material in the drop of 70% alcohol. This drives out the air trapped between the hyphae.
7. Tease out the material very gently with mounted needles*. Do not forget to sterilize the inoculating wire and the needles after use by heating to red heat in a Bunsen flame
8. Before the alcohol dries out add one or at most two drops of the stain. A common fault is to add too much stain to the preparation.
9. Holding the coverslip between your index finger and thumb, touch one edge of the drop of stain with the edge of the coverslip.
10. Lower the coverslip gently onto the slide, trying to avoid air bubbles. Your preparation is now ready for examination.

Teasing the colony often disrupts the delicate fruiting structures of the filamentous molds, making it difficult in some instances to observe the characteristic spore arrangements or hyphal attachments necessary for definitive identification. In such cases, a transparency tape mount or microslide culture may be required.

Cellophane Tape Preparation

The cellophane or transparency tape method for microscopic examination of fungi is simple to perform, inexpensive and, rapid. It preserves the conidial arrangements of the more delicate filamentous molds and allows one to make an accurate identification.

1. Place a drop of lactophenol aniline blue stain on a microscope slide.
2. Press the sticky side of unfrosted, clear cellophane tape gently but firmly to the surface of the colony, picking up a portion of the aerial mycelium.
3. Stick one end of the tape to the surface of the slide adjacent to the drop of stain.
4. Stretch the tape over the stain, gently lowering it so that the mycelium becomes permeated with stain.
5. Pull the tape taught and stick the opposite end to the glass, avoiding as much as possible the trapping of air bubbles.
6. Your preparation is now ready for examination.

Examination

Figure: Mucor species in LPCB mount (Image source)

1. Make the initial examination using a low-power objective lens. The thinner parts of the preparation, generally around the edges of the mounting material, will yield the best images.
2. Switch to a higher power 40X objective for a more detailed examination of spores and other structures.

What to Look For: Key Morphological Features Under LPCB

LPCB stains chitin in fungal cell walls blue, revealing the following structures:

Hyphae:

• Septate hyphae — hyphae divided by cross-walls (septa) into compartments. Seen in most medically important moulds: Aspergillus, Fusarium, Dermatophytes. Septa are often visible as faint lines perpendicular to the hyphal wall.
• Aseptate (non-septate) hyphae — hyphae without cross-walls; broad, ribbon-like, occasionally collapsing. Characteristic of Mucorales (Mucor, Rhizopus, Lichtheimia). Width is typically 6–25 μm — dramatically wider than septate mould hyphae (2–4 μm).
• Pseudohyphae — chains of elongated yeast cells that resemble hyphae but have constrictions at junctions. Characteristic of Candida albicans.

Key organisms and their LPCB appearance:

The three most important patterns to recognise immediately:

1. Broad aseptate hyphae → Mucorales → urgent clinical situation in immunocompromised patients
2. Septate hyphae with acute-angle branching + blue-grey conidia → Aspergillus fumigatus
3. Sickle-shaped macroconidia → Fusarium → important to distinguish from Aspergillus as treatment differs

LPCB vs Calcofluor White — When to Use Each

Both LPCB and calcofluor white stain fungal cell walls, but they have different applications:

Practical guidance:

• Culture identification → LPCB (standard choice — bright-field, full morphological detail)
• Direct specimen (skin scraping, BAL, nail, hair) → Calcofluor white if fluorescence microscope available; KOH mount if not
• Both can be used together for difficult cases where standard bright-field and fluorescence complement each other

For calcofluor white staining procedure, see: Calcofluor White Staining

LPCB for Intestinal Parasite Detection

The existing reference (Parija et al., 2003) notes a use that many microbiologists overlook: LPCB wet mounts of stool specimens can detect Cryptosporidium, Cyclospora, and Isospora oocysts.

Appearance on LPCB:

• Cryptosporidium parvum oocysts — small (4–6 μm), round, pink-tinged or pale against blue background; internal sporozoites occasionally visible
• Cyclospora cayetanensis oocysts — larger (8–10 μm), round, wrinkled appearance; appear as two clusters of sporocysts when sporulated
• Isospora belli oocysts — elongated oval (20–33 μm × 10–19 μm); most easily identified by size and shape

Important limitation: LPCB is not the preferred method for parasite detection — modified Ziehl-Neelsen (cold acid-fast stain) is the standard for Cryptosporidium and Cyclospora, and saline wet mount for intestinal parasites broadly. LPCB can be used when these dedicated methods are unavailable, but sensitivity is lower.

Troubleshooting LPCB Preparations

The transparency tape method is specifically recommended when fruiting structures have been disrupted by the tease mount — always attempt the tape method first for Aspergillus, Penicillium, and Fusarium where conidial arrangement is the key identification feature.

How to Remember: LPCB

The four components — "Please Let Cotton Grow":

• Phenol — kills
• Lactic acid — clears and preserves
• Cotton blue — stains chitin blue
• Glycerol — prevents drying

The broad vs narrow hyphal rule:

• Broad + aseptate = Mucorales (Mucor, Rhizopus) — emergency in diabetics and immunocompromised patients
• Narrow + septate = everything else (Aspergillus, Fusarium, dermatophytes)

This single distinction is the most urgent clinical differentiation in the mycology lab.

The three conidiophore shapes to know:

• Aspergillus = flask on a stick (conidiophore + vesicle + phialides)
• Penicillium = paintbrush (branching phialides)
• Fusarium = sickle/banana (curved macroconidia)

Composition of LPCB mount

Preparation of LPCB mount

LPCB can be purchased from commercial suppliers or prepared in-house by mixing the above-mentioned ingredients as mentioned in the steps below.

1. Add 20 mL of lactic acid in a beaker.
2. Add 40 mL of glycerol or glycerine.
3. Add 20 mL of distilled water
4. Add 22 g. of phenol crystals (22 ml of melted phenol).
5. Add 0.05 g. aniline blue.
6. Heat and stir the solution to dissolve the stain. Do not boil or go over 100°C.
7. Mix well and let the solution cool. You can store the solution at room temperature and dispensed it with a pipette when needed.

Key Exam Facts in One Table

References

1. Winn, W. C., & Koneman, E. W. (2006). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Color Atlas & Textbook of Diagnostic Microbiology). Lippincott Williams & Wilkins
2. Parija, S. C., Shivaprakash, M. R., & Jayakeerthi, S. R. (2003). Evaluation of lacto-phenol cotton blue (LPCB) for detection of Cryptosporidium, Cyclospora and Isospora in the wet mount preparation of stool. Acta tropica, 85(3), 349–354. https://doi.org/10.1016/s0001-706x(02)00265-6
3. Khubnani, H., Sivarajan, K., & Khubnani, A. H. (1998). Application of lactophenol cotton blue for identification and preservation of intestinal parasites in faecal wet mounts. Indian journal of pathology & microbiology, 41(2), 157–162.
4. Larone DH. Larone's Medically Important Fungi: A Guide to Identification. 6th ed. ASM Press; 2018.
5. Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology. 9th ed. Elsevier; 2020.